US9631169B2 - Three dimensional tissues for high-throughput assays - Google Patents

Three dimensional tissues for high-throughput assays Download PDF

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US9631169B2
US9631169B2 US12/996,168 US99616809A US9631169B2 US 9631169 B2 US9631169 B2 US 9631169B2 US 99616809 A US99616809 A US 99616809A US 9631169 B2 US9631169 B2 US 9631169B2
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tissue
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US20110118143A1 (en
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Tetsuro Wakatsuki
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Invivo Sciences Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/06—Methods of screening libraries by measuring effects on living organisms, tissues or cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms

Definitions

  • cell-based assays have been developed for monitoring cellular activities and functions such as cell viability, cell proliferation, gene expression, and intra- and intercellular signaling. Such assays are useful for high throughput (HTP) screening applications.
  • HTP high throughput
  • High throughput cellular assay systems using a three dimensional tissue model and methods of performing such assays are provided herein.
  • the assays are useful for monitoring the response of the tissue to treatment with a variety of agents and stressors.
  • Three-dimensional tissues are formed on a scaffold positioned within a well in a multi-well plate. The tissues are suspended above the bottom of the well. An assay is performed on the suspended tissue and the output of the assay is measured in the well.
  • methods of detecting the response of a tissue to an agent include contacting a bio-artificial tissue with an agent, performing an assay which produces an indicator using the bio-artificial tissue, and detecting a level of the indicator in the well.
  • the level of the indicator is indicative of the response of the bio-artificial tissue to the agent.
  • the bio-artificial tissue used in the assay comprises cells and extracellular matrix and is formed on a scaffold support without a fastener to facilitate tissue adhesion.
  • the scaffold support is positioned above the bottom of a well.
  • the output of the assay may be a colorimetric or fluorescent product, and the accumulation of the product may be measured using a plate reader.
  • Exemplary assays that may be carried out with the systems in accordance with the invention include, but are not limited to, cell proliferation assays, cell death assays, apoptosis assays, protein expression assays, gene expression assays, enzymatic assays, signaling assays such as kinase activity assays, Ca2+ signaling assays and GPCR signaling assays, assays to assess mitochondrial activity, and extracellular matrix degradation assays.
  • FIG. 1 a shows a high throughput system illustrating use of triangular and rectangular (alternative shape) scaffolds shown in FIG. 1 b , made of stainless steel wire about one millimeter in diameter, which provide supports upon which reconstituted three dimensional tissues form.
  • FIG. 2 shows several views of the scaffold.
  • FIG. 2 a is a top elevation view of the scaffolds.
  • FIG. 2 b is a side elevation view of the scaffold.
  • FIG. 2 c is a side elevation view of one way of connecting several scaffolds to each other for ease of use in high throughput applications.
  • FIG. 3 is a photograph showing a typical engineered three-dimensional tissue.
  • FIG. 4 is a diagram depicting the distinction between a three dimensional tissue and a monolayer of cells.
  • the graphs demonstrate the increased cell-based assay signal strength that may be realized by using a three-dimensional tissue instead of a cellular monolayer.
  • FIG. 5(A) is a photograph of The PalpatorTM.
  • the system includes an isometric force transducer (a), probe baths (b), and a hydrogel tissue construct (HTC) stage (c) on a temperature regulator plate (d).
  • FIG. 5(B) is a photograph showing the x-y-z motion robotic arm which positions the force transducer with attached probe above the HTCs and lowers the probe onto the tissue for force measurement.
  • FIG. 5(C) is a schematic of the robotic arm with attached force transducer and probe.
  • FIG. 5(D) is a photograph of the probe positioned 1 mm above the mid-plane of a HTC (a) and then lowered until it first touches (b) and then stretches (c) the tissue.
  • FIGS. 5(E) is a graph showing representative forces recorded during HTC indentation. Arrows a, b, and c show force measurements associated with probe positions shown in (D).
  • FIGS. 5 ( f and g ) are graphs showing that Rho kinase inhibitor 1 (RKI1), Cytochalasin D (CD), rotenone (ROT), and 2,4-dinitrophenol (DNP) dose-dependently reduced HTC contractile force.
  • HTCs were preconditioned, then treated with varying concentrations of the four drugs as indicated. Forces were measured at 3 (f) and 24 hr (g). Force measurements were expressed relative to control, medium treated, HTCs (F c ).
  • FIG. 5( h ) is a graph showing that Cytochalasin D treatment reduced cellular F-actin.
  • HTCs treated with drugs for 24 hours were fixed and labeled with Alexa 568 conjugated phalloidin. Fluorescence intensity was read on a plate reader and plotted relative to control, media treated, HTCs (I c ).
  • Data show mean and SEM of 3 replicates; @ p ⁇ 0.02 by Student's t-test vs. controls (Ctrl).
  • FIG. 6( c ) is a graph showing a representative tracing of microscopically quantified TMRE signal in REF monolayers treated with DNP.
  • FIG. 6( d ) is a graph showing a representative tracing of microscopically quantified TMRE signal in the bottom cell layer of a HTC treated with DNP.
  • FIGS. 6 ( e and f ) are graphs showing that DNP dose-dependently uncoupled HTC mitochondrial potential.
  • HTCs preloaded with TMRE 100 nM for 30 min) were treated with varying concentrations of CD, RKI1, DNP, and ROT.
  • TMRE fluorescence signal was measured using a plate reader at 3 (a) and 24 (b) hr post treatment. Signal intensity was expressed relative to the control, medium treated, HTCs (I c ). The mean and SEM of 4 (several with 2 or 3) replicates are shown; Z-factor>0.64 ( # ) and 0.46 (*).
  • FIG. 7( a ) is a graph showing that CD and ROT exhibited dose-dependent cytotoxicity. Viability of drug treated HTCs was determined by MTT assay at 24 hours. Absorbance of formazan (converted from MTT in viable cells) was read on a plate reader and expressed relative to control, medium treated, HTCs (A C ). 10% DMSO treatment was used as a positive control for this assay which yielded A/A C of 0.11 ⁇ 9 ⁇ 10 ⁇ 3 (not shown in graph); Z-factor>0.85 for control vs. 10% DMSO analysis. Data show mean and SEM of predominately 4, with several 2 and 3, replicates.
  • FIG. 7( b ) is a work flow schematic of HTC screening.
  • FIG. 7 ( c - f ) are graphs showing phenotypic profiles used to screen the compounds.
  • Physiology data i.e., tissue force (Force), mitochondrial potential (Mit.
  • the usefulness of many of the available cell-based high throughput screening assays has been limited due to the low signal strength generated by colorimetric or fluorescent indicators.
  • Such assays generally use cells in a monolayer or suspension culture, rather than cells present in a tissue.
  • monolayers and suspended cells do not necessarily behave like cells in vivo in the three-dimensional environment of a tissue.
  • the assays and tissues presented here allow these high throughput assays to be performed in a tissue-based system which more closely resembles an in vivo setting.
  • the signal generated as an output by an assay on a three-dimensional tissue is amplified.
  • the invention improves the efficiencies of measuring cell physiology for assays using optical detection systems (such as spectrophotometric and fluorescence plate readers).
  • This is an additional feature of a physiology profiling system using tissues, such as engineered or bio-artificial tissues.
  • engineered tissues biological-artificial tissues
  • cells growing in the engineered tissues (3D tissue organoids) may be loaded with fluorescent probes that serve to report on the physiology of intra- and/or extra-cellular activities.
  • the fluorescence probes report the physiological states by changing their intensities or shifting their emission spectra.
  • the methods include contacting a bio-artificial tissue with an agent, performing an assay that produces an indicator on the bio-artificial tissue, and detecting the level of the indicator in the well.
  • the level of the indicator is indicative of the response of the bio-artificial tissue to the agent.
  • the bio-artificial tissue used in the assay comprises cells and extracellular matrix and is formed on a scaffold support without a fastener to facilitate tissue adhesion.
  • the assays may be adapted for high throughput screening methods.
  • the bio-artificial tissue may be contacted with an agent via any means available to those skilled in the art.
  • the agent may be added to a well containing the bio-artificial tissue or may be provided to the cells prior to forming the bio-artificial tissue.
  • the agent may be brought into contact with the cells by means of a vector, such as a viral vector or liposome, or via receptor-mediated targeting.
  • Bioartificial tissue models can be used to assess quantitatively and rapidly the effects of many different classes of agents, including but not limited to, pharmaceuticals or potential pharmaceuticals, toxins, chemicals, nucleic acids, peptides, polypeptides and microorganisms, including pathogens or vectors.
  • agents useful as activators include, but are not limited to, fetal bovine serum (FBS), lysophosphatidic Acid (LPA); thrombin, growth factors including epidermal growth factor (EGF), platelet derived growth factor (PDGF), angotensin-II, endothelin-1, vasopressin and combinations thereof.
  • FBS fetal bovine serum
  • LPA lysophosphatidic Acid
  • thrombin growth factors including epidermal growth factor (EGF), platelet derived growth factor (PDGF), angotensin-II, endothelin-1, vasopressin and combinations thereof.
  • Inhibitors include, but are not limited to, inhibitors which bind cell surface receptors including a receptor antagonist for angiotensin II receptor and also inhibitors that act within the cell.
  • Inhibitors useful herein include, but are not limited to, those which inhibit signal transduction pathways including genistein, herbimycin and agents which act on the cytoskeleton.
  • Inhibitors also include, but are not limited to, cytochalasin D, latrunclin B, paclitoxol, nocodazole, calyculin A, butane-dione-monoxime (BDM) and combinations thereof.
  • the amount of agent(s) provided to the bio-artificial tissue is an amount effective to elicit a response from or by a tissue model.
  • An effective amount is generally between about 1 nM to 100 mM, suitably 100 nM to 1 mM, more suitably 500 nM to 500 ⁇ M.
  • an assay can be performed on the bio-artificial tissue.
  • Any assay which can be designed to produce an indicator, such as a colorimetric, fluorescent or radioactive indicator, can be adapted for use with the 3D bio-artificial tissues in accordance with the invention.
  • Assays including but not limited to cell proliferation assays, cell death assays, apoptosis assays, protein expression assays, gene expression assays, enzymatic assays, signaling assays such as kinase activity assays, Ca2+ signaling assays and GPCR signaling assays, assays to assess mitochondrial activity, and extracellular matrix degradation assays may be used in the methods described herein.
  • Assays developed for use with monolayers of cells in particular those dependent upon uptake of agents or assay reagents by cells, will require longer incubation times in order to allow the agents and assay reagents to be taken up by the cells within the bio-artificial tissue. Assay reagent concentrations will also require adjustment.
  • the level of indicator produced by the assay refers to the output of the assay or the signal resulting from performance of the assay.
  • the level may relate to an amount of indicator produced or an alteration in the indicator itself, for example a change in emission spectra, or uptake of a labeled indicator by the cells.
  • the level of the indicator is indicative of the response of the bio-artificial tissue to the agent. Detection of the level of the indicator may make use of microscopes, optical or fluorescent plate readers or scintillators, dependent on the indicator and assay chosen. The sensitivity of signals detected by microscopes is higher than that of plate readers. Microscopes focus on a cell layer to collect optical signals very efficiently (the detection volume is focused and small).
  • Plate readers use a diffused (unfocused) optical beam (e.g., unfocused laser) to detect molecules in a large volume illuminated by the beam.
  • the plate reader measures optical signals from a single cell layer.
  • cells in the engineered tissues form at least 5-10 layers, therefore the plate reader can measure a large number of cells at once resulting in optical signals detected by the plate readers that are amplified at least 5-10 fold. See FIG. 4 .
  • Plate readers can read signals much faster because they do not require focusing on individual cells, therefore use of plate readers is suitable for high throughput applications.
  • the analyses of images taken by microscopes are also more time-consuming than those taken using a plate reader.
  • a plate reader can be used to obtain the same information (e.g. Ca concentration), but the signal is much weaker than that obtained by the automated microscope.
  • One solution to this problem is to increase the number of cells measured by the plate readers. Cells form multiple layers in the engineered 3D tissues, therefore the plate reader can measure more cells in the same area.
  • Plate readers are superior to microscopes for analyzing the dynamic properties of live-cells. Improved signals and high statistical significance of measurements using the tissue constructs as disclosed herein will allow application of many small scale assays to cell-based high content analysis. Currently microscopes are generally used due to the increased sensitivity, but the methods described herein will allow plate readers to be used for cell-based high content analysis.
  • FIGS. 1 a and 1 b in which a scaffold 20 is shown, suitably including a frame 22 , e.g., a triangular frame.
  • a reconstituted tissue 26 forms on scaffold 20 .
  • wells 42 are slightly tapered toward the bottom and are wells of a 96-well plate 40 .
  • the scaffold 20 is securely positioned above the bottom of each well 42 , suitably about 1 mm.
  • a non-polymerized solution of collagen containing cells and appropriate cell culture media as described is poured into the wells, filling them to a level 3 mm above the bottom of the well ( FIG. 1 a ).
  • the 96-well plate 40 may be incubated at 37° C. with 5% CO 2 .
  • the cells self-assemble into the bio-artificial tissue 26 and compress the collagen matrix by squeezing out liquid thereby reducing the total volume by about ten fold.
  • the reconstituted or bio-artificial tissue contracts into a small sphere floating in the tissue culture medium.
  • Scaffolds 20 are suitably made of any non-porous, bio-compatible material, such as metal, nonmetal, or plastic.
  • the scaffold was made of stainless steel.
  • materials including, but not limited to, glass, polypropylene or polystyrene may also be suitably used to produce the scaffold.
  • Frame 22 is suitably supported above the bottom 43 of well 42 .
  • Frame 22 may be supported by the side of the well by using tissue culture plates 40 with tapered wells.
  • frame 22 may be supported above the bottom of well 42 by using specially designed plates 40 with built-in scaffolds attached to the side of the well or with wells having ledges on which frame 22 rests.
  • scaffold 20 may include at least one leg 24 attached to frame 22 to support the frame above the bottom of the well. The number of legs 24 required to support the frame will vary depending on the shape of the frame.
  • FIG. 1 b depicts scaffold 20 with 4 legs, but scaffolds may be designed with fewer or more legs as depicted in FIG. 2 .
  • Legs 24 may be used to support frames 22 by projecting down from the frame and touching the bottom of well 42 or legs 24 may project upwards from frame 22 and support the frame of scaffold 20 by anchoring the scaffold to the top 45 of well 42 .
  • leg 24 may have a small hook structure at the end that allows scaffold 20 to hang from the top of the well ( FIG. 2( b ) ).
  • frame 22 of scaffold 20 is suitably supported above the bottom of well 42 , the exact distance is not critical as long as the tissue can be bathed in media.
  • frame 22 is at least about 0.25 mm above the bottom of the well, more suitably the frame is at least about 0.5 or 1.0 mm above the bottom of the well.
  • Scaffold 20 may take a wide array of shapes.
  • the collagen-containing matrix can be compressed into different shapes using different scaffold shapes such as a circle or rectangle as depicted in FIG. 2 .
  • Other scaffold shapes such as those shown in FIG. 1 b and FIG. 2 , produced tissue strips with different widths and shapes.
  • Any shape scaffold 20 can be used, including but not limited to, circular, rectangular, triangular, pentagonal, hexagonal, or other higher order polygons.
  • the scaffold may also be formed of more than one member.
  • scaffold 22 may be formed of two parallel members spaced apart with or without one or more perpendicular member connecting them ( FIG. 1 b and FIG. 2 ).
  • cells self-assemble to form a tissue model conforming to the shape of the scaffold, i.e., support, for example a wire frame.
  • the tissue overlays the members of the scaffold and spans the space between the members.
  • the cells form a membrane spanning among the three edges, which is illustrated in FIG. 1 a .
  • the scaffold in the Examples was made of members having about 1 mm cross-sectional diameter, but scaffolds may suitably have smaller or larger cross-sectional diameters.
  • the scaffold is made up of one or more members with cross-sectional diameters between about 100 ⁇ m and about 2 mm.
  • the scaffold is comprised of generally cylindrical or tubular members that allow the tissue to form around the members such that the tissue overlays the members.
  • the members comprising the scaffold are suitably somewhat rounded to minimize ripping of the tissue when a force is applied.
  • members with a rectangular cross-section could be utilized if the edges were rounded such that the tissue did not tear when force was applied.
  • the members are suitably made of a non-porous material and have a cross-sectional diameter of less than about 2 mm, suitably about 1 mm.
  • the bio-artificial tissue forms a membrane structure spanning a horizontal cross-sectional space between or across the members comprising the scaffold.
  • the horizontal cross-sectional space that the bio-artificial tissue spans is suitably larger than 10 ⁇ m, but can be as large as the well 42 allows, suitably the tissue spans a space between about 100 ⁇ m and about 5 mm, more suitably between 1 mm and 4 mm.
  • a typical bio-artificial tissue depicted in FIG. 3 is approximately 4 ⁇ 4 ⁇ 0.8 mm and formed in an 8 mm ⁇ 8 mm square chamber. (The shape of chamber was modified for viewing the sample. The tissue was fixed with formaldehyde (10%) and stained with orange dye for clear viewing).
  • FIG. 3 depicts a prototype multi-well plate 40 comprising scaffolds 20 .
  • an 8-well plate was machined from a polycarbonate bar (25 ⁇ 60 ⁇ 10 mm) using a tabletop CNC mill (Sherline Products Inc., Vista, Calif.).
  • the 8 square wells 42 of 8 ⁇ 8 mm contained 2 stainless steel bars that make the frame 22 (1 mm diameter). The centers of the stainless steel bars were located 2 mm above the bottom of the well and 2 mm from the side of the well such that the 2 bars were 4 mm apart.
  • a microscope coverslip No. 1 thickness, Fisherbrand was used to seal the bottom of each well using silicon glue (Dow Chemical Co., Midland, Mich.) to facilitate microscopic imaging.
  • scaffolds 20 may be joined together by a connector 28 in groups including but not limited to, 2, 4, 8, 12 or 96 scaffolds as depicted in FIG. 2C .
  • Connectors 28 may be made to be readily separable, e.g., such that a quick tugging motion will break the connection and allow the user to customize the number of scaffolds used.
  • scaffolds and bio-artificial tissue system described herein may also be adopted for use by one of skill in the art in any multi-well plate, including but not limited to, 6 well, 8 well, 12 well, 24 well, 48 well, 192 well or 384 well plates.
  • a porous support material, or other fastener, such as a Velcro fastener is not needed to facilitate tissue adhesion even to the non-porous stainless steel surfaces of the wire frame used.
  • the collagen was compressed to a greater extent at the outer portion of the membrane or tissue strip and allowed the tissue to be suspended on the scaffold without the need for a fastener. Therefore, this outer portion of the membrane can withstand the stress produced by the cells and prevents ripping the bio-artificial tissue off from the wire frame.
  • the bio-artificial tissue in accordance with several embodiments of the invention provides a three-dimensional tissue which is more akin to an in vivo setting than the cell monolayers of current cell-based assays.
  • the increased cell numbers result in increased signal output for a specific test assay.
  • the invention thus provides a mechanism by which all manner of cell-based assays can be successfully generated in a high throughput system.
  • the tissue unlike other three-dimensional tissues the tissue here is not grown on a mesh or frame which then interferes with optical measurement. Instead the tissue spans the frame and optical measurements are possible.
  • the system of the invention not only uses smaller amounts of reagents due to the small size of the tissues required for testing, but also allows analysis of tissues maintained in tissue culture conditions, including maintenance of constant temperature and sterile conditions throughout the assay procedure.
  • assays may be performed in a laminar flow hood to avoid contamination of the bio-artificial tissues.
  • the cells within the tissue are stable such that the assays can be repeated on the same set of bio-artificial tissues several times over the course of hours, days, or even weeks.
  • Multi-well plate 40 may be a specially designed plate comprising scaffolds 20 for holding the bio-artificial tissues or suitably may be a generally commercially available tissue culture multi-well plate to which scaffolds may be added.
  • the number of wells per plate may vary. Typically plates with between 2 and 1000 wells will be used, suitably plates with between 50 and 500 wells will be used.
  • the cells used to form the bio-artificial tissues may include, but are not limited to muscle cells, endothelial cells, epithelial cells, fibroblasts, embryonic stem cells, mesenchymal stem cells and cardiac cells.
  • the bio-artificial tissue may comprise cells and collagen or cells and extracellular matrix.
  • Collagens useful in formation of bio-artificial tissues include collagen Classes 1-4 which include all Types I-XIII and combinations thereof.
  • extracellular matrix may also be used in formation of bio-artificial tissues, such as hydrogels or Matrigel®.
  • the cells in the reconstituted tissue models in accordance with several embodiments of the invention are in an environment that resembles their condition in natural tissues and organs. Therefore, results of the assays using this method yield results similar to those obtained using animal models. It is contemplated that some of the animal testing can be replaced by using tissue models in accordance with the invention. For example, some tests of agents acting on skin can be conducted using artificial living tissues.
  • a triangular frame made of stainless steel wire 1 mm in diameter was employed as a scaffold on which the reconstituted tissue formed.
  • the wells are slightly tapered toward the bottom and the frame is securely positioned 1 mm above the bottom of the well ( FIG. 1 a ).
  • a non-polymerized solution of collagen containing cells and appropriate cell culture media was poured into the wells filling the wells to a level 3 mm above the bottom ( FIGS. 1 b and 3 ).
  • the 8-well plate in FIG. 3 was incubated at 37° C. with 5% CO 2 . During the incubation, cells compressed collagen matrices by squeezing liquid out from the porous collagen matrix.
  • the reconstituted tissue contracted into a small sphere floating in the tissue culture medium.
  • the collagen matrix was compressed into shapes corresponding to shapes of the frames.
  • a triangular wire frame made a membrane spanning among the three edges as shown in FIG. 1 a .
  • Other wire frame shapes such as one shown in FIG. 1 b , produced tissue strips with different widths.
  • a porous support material such as a Velcro fastener was not required to facilitate tissue adhesion even to the non-porous stainless steel surfaces of a wire.
  • the collagen was compressed to a greater extent at the outer portion of the membrane or strip. Therefore, this outer portion of the membrane can withstand stress produced by the cells and prevented it from ripping the membrane off the wire frame.
  • Rat embryonic fibroblasts (REF-52) were cultured in Dulbecco's Modified Eagle's Medium (DMEM, MT10013CM, Fisher Scientific, Pittsburgh, Pa.) supplemented with 10% fetal bovine serum (FBS, S11050, Atlanta Biologicals, Lawrenceville, Ga.). Cells were sub-cultured every two to three days.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • HTCs hydrogel tissue constructs
  • REF-52 cells between passages 40 to 70
  • the trypsin solution was decanted and the cell pellet was re-suspended in 10% FBS DMEM medium. This cell suspension was diluted in HTC tissue solution to achieve a final concentration of 8 ⁇ 10 5 cells per ml.
  • the HTC tissue solution consisted of 10% FBS DMEM, 1 mg/ml of type 1 collagen (354249, BD Biosciences, San Jose, Calif.) in 0.02 N acetic acid, sufficient sodium hydroxide to neutralize the acid in the collagen, and sufficient 5 ⁇ DMEM to compensate for the volume of collagen and NaOH.
  • the HTC tissue solution was kept on ice until its distribution into our custom-made tissue molds ( FIG. 3 ). Each mold contains 8 separate HTC-forming wells with two built-in horizontal support bars.
  • the PalpatorTM (as described in U.S. Pat. No. 7,449,306, which is incorporated herein by reference in its entirety) was used to quantify the contractility of the HTCs.
  • the molds were placed on the stage of the PalpatorTM which automatically inserted a probe into each well and stretched the individual HTC.
  • the probe was connected to a force transducer which measured the resistance force induced in the HTC in response to stretch and exported the values to a computer for recording.
  • a custom Matlab algorithm was used to process and analyze the force data to report a numerical parameter that is indicative of the active cell force in the HTC.
  • HTCs at 24 hr post treatment were always pre-conditioned before force measurement.
  • TMRE tetramethylrhodamine
  • 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT, M-6494, Invitrogen) was used to quantify cell viability in the HTCs.
  • MTT was added to each well to achieve 0.5 mg/ml. MTT was left in the wells for 2 hr and then removed.
  • the formazan dye that forms in the cells were then suspended in 500 ⁇ l of isopropanol (S77795, Fisher Scientific) containing 0.1 N hydrochloric acid. Two hundred microliters of the formazan solution is then put into a 96-well plate well and read on the Synergy HT plate reader. Absorbance at 570 and 650 nm were recorded and the difference (A 570 -A 650 ) was used as the absorbance intensity of the formazan.
  • DNP was diluted to 1 M in DMSO and then to 100, 10, and 1 mM in ⁇ F/G/P DMEM. Following HTC preconditioning and background force (i.e. pre-drug) measurement, 50 ⁇ l of the appropriate drug dilutions were added to each well to achieve the desired treatment concentration. Fifty microliters of ⁇ F/G/P DMEM was added to the control wells. Upon drug addition, medium in the wells was mixed by pipetting four times.
  • REF-52 cells were plated on 35 mm culture dishes (50,000 cells) in 10% FBS medium (2 ml). Cells are incubated over night and then treated with compounds. Concentrated CD, RKI1, DNP, and ROT were dissolved in ⁇ F/G/P medium and then diluted 10 ⁇ into each plate of cells (220 ⁇ l per 2 ml medium). Cells were incubated with drugs for 24 hours prior to fixing. To fix, treated cells were rinsed once with 2 ml of phosphate buffered saline (PBS, D5652) and then incubated in 1 ml of 4% paraformaldehyde (Sigma) solution (in PBS) for 30 min. Fixed cells are rinsed twice and then stored in 2 ml of PBS.
  • PBS phosphate buffered saline
  • Labeled cells were mounted with Vectashield (Vector Laboratories, Burlingame, Calif.), covered with a cover slide and then sealed with nail polish. The plate was then inverted on to a Leica SP5 confocal microscope (Leica Microsystems, Bannockburn, Ill.) and imaged with 63 ⁇ water immersion objective. Alexa 568 was excited using the 543 laser line and DAPI was excited using a MaiTai multi-photon laser.
  • HTCs hydrogel tissue constructs
  • PalpatorTM FIG. 5 a , 5 b , 5 c
  • the 3D HTCs provides a more natural microenvironment and the cells can better mimic in vivo morphology and physiology.
  • the self-supporting HTCs can be stretched using a force probe for measuring cellular mechanics ( FIG. 6 d ).
  • the HTCs' mechanical properties can be quantitatively measured ( FIG. 6 e ) without cell labeling, sophisticated microscopy, or image analysis.
  • Tissue force is maintained through the integrity of the cellular cytoskeleton, especially actin and myosin.
  • the HTCs were stained with Alexa 546 conjugated phalloidin.
  • the intensity of Alexa-phalloidin was measured by a plate reader and F-actin content in HTCs treated with CD (2 ⁇ M) was significantly reduced ( FIG. 5 h ). This is in agreement with our previous report that CD mediated reduction in tissue force was due to the loss of intact F-actin.
  • force reductions by H1152 and DNP were not related to the loss of F-actin. Alexa-phalloidin labeling in these HTCs were comparable to controls.
  • F-actin was also reduced by ROT treatment; however this result was inconclusive due to insufficient statistical significance.
  • Microscopic analysis of phalloidin-stained cells treated with 2 ⁇ M CD exhibited extensive disruption of F-actin as early as 3 hours ( FIG. 5 j ). By 24 hours, short F-actins were re-distributed within less-spread ( FIG. 5 o ) and binuclear ( FIG. 5 t ) cells.
  • RKI, DNP, and ROT did not dramatically affect F-actin morphology ( FIG. 5 k - m ).
  • RKI treated cells did exhibit limited membrane ruffling and reduced phalloidin staining in the central region of the cells ( FIG. 5 c ).
  • Confluency of cells treated with DNP ( FIG. 5 q ) and ROT ( FIG. 5 r ) for 24 hours were less than that of control ( FIG. 5 n ).
  • TMRE tetramethylrhodamine ethyl ester
  • HTC experiments still showed superior sensitivity in detecting the dose-dependent MMP reduction by DNP over cell-monolayer experiments. Considering the superior sensitivity of signal detection, improved signal-to-noise ratio of the data, and the compatibility of plate reader scanning to HTP applications, we used HTCs for studying the compounds' effects on MMP.
  • the observed level of CD toxicity was similar to the reported LD 50 of 5-30 ⁇ M in human epidermoid cell lines. Higher ROT concentration, 10 ⁇ M, was required to reduce MTT signal by 40% ( FIG. 7 a ). This level of ROT toxicity is slightly higher than the 25% toxicity previously reported in HepG2 cells. RKI and DNP treatments for 24 hours did not affect MTT signal.
  • FIG. 7 b A diagram of the integrated screening workflow shows the efficiency in obtaining high-content physiological information using HTCs.
  • the results were summarized as panels of phenotypic profiles that represent the physiological impact of the compounds ( FIG. 7 c - f ).
  • DNP, CD, and RKI were all very effective at reducing HTC force.
  • DNP exhibited extensive uncoupling effect of mitochondrial potential ( FIG. 7 c ) while CD was highly toxic ( FIG. 7 d ).
  • ROT on the other hand, had a moderate negative impact on HTC force, mitochondrial potential, and viability ( FIG. 7 f ).
  • RKI was identified to be the best candidate compound with force-reducing EC 50 of 0.1 ⁇ M with limited mitochondrial and viability toxicity. This result was not surprising since RKIs are well known to be cardioprotective, non-toxic, compounds that can effectively reduce tissue stiffness. Further, an RKI drug, Fasudil, has recently completed phase II clinical study for atherosclerosis and hypercholesterolemia.
  • the inventors have demonstrated how HTCs can be used to screen for compounds that can reduce tissue contractile force and yet have minimal effects on mitochondrial functions and cellular viability.
  • This HTC-based screening system we were able to study cellular physiology and quantify mechanical force within a more natural microenvironment, i.e., embedded in a three-dimensional matrix structure, as compared to two-dimensional cultures.
  • the compact and multi-layered arrangement of cells in the HTCs greatly increased the detection limit and the signal-to-noise ratio of fluorescent assays.
  • Z-factors ranging from 0.44 to 0.85, the high accuracy and robustness of the assays will facilitate the incorporation of HTCs into existing HTP screening workflow.
  • the inventors provide a high throughput system utilizing a three dimensional tissue model for performing cell-based assays.
  • the foregoing description is considered as illustrative only of the principles of the invention. Further, since numerous modifications and changes may readily occur to those skilled in the art, it is not intended to limit the invention to the exact construction and operation shown and described, and accordingly, all modifications and equivalents are considered as falling within the scope of the invention.
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